PROJECT SUMMARY The goal of this proposal is to test whether the biology of glycosphingolipid trafficking can be harnessed for delivery of therapeutic peptides and antigens across tight mucosal barriers in vivo. Currently, there are no effective methods for oral drug delivery across tight mucosal epithelial barriers lining the intestine. Here, we propose studies towards the development of a lipid-based platform for mucosal delivery of therapeutic peptides and antigens exploiting an endogenous glycoshingolipid-based trafficking pathway across polarized epithelial cells. This pathway sorts short- or unsaturated GM1-ceramides across the cell to the basolateral membrane - thus breeching the epithelial barrier by transcytosis. Our aim is to design GM1 carriers that internalize therapeutic cargoes and efficiently cross the intestinal epithelial barrier. We have recently identified GM1 species that are efficiently released from cell membranes after transcytosis and another set of GM1 species that are not efficiently released, but are rather retained on membranes for longer periods. We will expand our proof of principle studies by mucosal administration of the non-native GM1 species fused to the incretin hormone GLP-1, as model for treatment of Type II Diabetes. Further structure-function studies of the extracellular oligosaccharide domain of GM1 and mechanistic studies to enable the technology are also a focus of the current proposal. The GM1 platform would be applicable to the mucosal delivery of a wide range of large and small molecule drugs and therapeutic proteins and peptides- including mucosal antigens for immune system modulation. I will then test if GM1 species can allow for delivery of antigens (modeled by ovalbumin) across mucosal barriers such as enabling induction of immunological tolerance (or mucosal vaccination). I will leverage our unique reagents and research approach, the available resources throughout the Lencer Lab and Longwood Medical Campus, and group of expert scientists we have already assembled to complete the proposed experiments and develop my career as an independent researcher and achieve my goal to make novel contributions to the field of lipid biology and vaccine development.